The major goal of this application is to understand molecular mechanisms of ventricular fibrillation (VF). We have developed a model of stable VF in the Langendorff-perfused guinea pig heart in which distinct patterns of organization develop in the left (LV) and right (RV) ventricles, and VF excitation frequencies are distributed throughout the ventricles in clearly demarcated domains. The highest frequency domains are always found on the anterior wall of the LV, demonstrating that a high frequency reentrant source (a rotor) that remains stationary in the LV is the mechanism that sustains VF in this model. Computer simulations predict that the inward rectifying potassium current (IK1) is an essential determinant of rotor stability and rotation frequency. In addition, our preliminary studies strongly suggest that the outward component of IK1 of cells in the LV is significantly larger than in the RV. Because of their ubiquitous expression in the heart and their strong rectification properties, Kir2.x channels are thought to play an essential role in shaping IK1 at potentials between -100 and 0 m V, with the weaker rectifying channel, TWIK-1 playing a role at more positive potentials. Thus, our general hypothesis is that differences in the expression and distribution of Kir2.x channels are responsible for LV vs. RV differences in outward IK1 density. We will use a combination of molecular, patch-clamp and optical mapping techniques to pursue the following Specific aims: 1. To characterize the molecular and electrophysiological properties of Kir2.x channels expressed in a mammalian cell line at physiologic temperature. 2. To characterize the role of individual Kir2.x channels in the guinea pig heart by determining whether they are differentially expressed in various regions of the heart; by recording single channel openings from isolated guinea pig ventricular myocytes; and using antisense oligonucleotides to determine the relative roles of each Kir2.x in IK1. 3. To characterize the role of individual Kir2.x channels in the dynamics of reentry, and determine the effects of manipulations of Kir2.x channel isoform expression on the dynamics of rotors and wave propagation in cultured cardiac cell monolayers and in the ventricles of the isolated guinea pig heart. The work outlined in this application should greatly increase our understanding of the molecular mechanisms underlying VF and may lead to new and safe approaches to prevent sudden cardiac death.